2odd protein, gene, vector, cell, composition and use thereof

Abstract

Disclosed in the present invention are an Fe(II) / α-ketoglutaric acid-dependent dioxygenase protein, a gene encoding Fe(II) / α-ketoglutaric acid-dependent dioxygenase, a recombinant vector, a transgenic cell, a composition, and the use thereof in the improvment of the herbicide resistance of a crop. Further disclosed is a method for improving the herbicide resistance of a crop. The Fe(II) / α-ketoglutaric acid-dependent dioxygenase and the gene encoding same in the present invention can be used for improving the resistance of a crop to an HPPD inhibitor.

Description

2ODD protein, gene, vector, cell, composition and application thereof Technical Field

[0001] The present invention relates to the field of genetic engineering, and in particular to a 2ODD protein, gene, vector, cell, composition and application thereof. Background Art

[0002] The rice gene HIS1, also known as 4-hydroxyphenylpyruvate dioxygenase inhibitor sensitive gene No. 1, encodes an Fe(II) / α-ketoglutarate-dependent dioxygenase (2ODD), which hydroxylates triketide HPPD inhibitors, conferring resistance to these inhibitors in rice. Furthermore, multiple HIS1 homologs, designated HSL (HIS1-Like), have been discovered in rice. HSL genes have also been found in other plants, including barley, wheat, sorghum, maize, and Arabidopsis.

[0003] Current research on HIS1 and HSL has revealed that the proteins encoded by HIS1 and its homologous gene, HSL, can catalyze the detoxification of triketone HPPD inhibitors, with the catalytic products having significantly reduced inhibitory activity against HPPD. Since HPPD is a key herbicide target, the ability of HIS1 and HSL to detoxify HPPD inhibitors could be exploited in the development of HPPD-resistant crops.

[0004] Currently, there are 13 major HPPD inhibitor herbicides on the market. Among the more than 20 herbicide categories globally, HPPD inhibitors have seen rapid growth in recent years, garnering significant industry attention. Their low resistance risk is the cornerstone of this category's leading growth. Given their superior product characteristics, HPPD inhibitors hold significant market potential for future growth.

[0005] However, HPPD inhibitor herbicides are primarily used in corn and wheat fields. They are not widely used in areas where cash crops such as peanuts, soybeans, rice, and sorghum are grown due to crop safety concerns. Furthermore, some highly active HPPD inhibitors currently in development have not been further developed due to a lack of adequate crop safety. Herbicide-resistant crops, such as glyphosate-resistant corn and soybeans, have brought significant benefits to farmers and the environment over the past 20 years.

[0006] Therefore, the development of new herbicide-resistant crops, such as crops resistant to HPPD inhibitor herbicides, has important theoretical significance and economic value.

[0007] Summary of the Invention

[0008] The purpose of the present invention is to overcome the defect of the existing HPPD inhibitor herbicides that the inhibitory activity and crop safety cannot be taken into account at the same time, and to provide a class of Fe(II) / α-ketoglutarate-dependent dioxygenase proteins resistant to HPPD inhibitors, their encoding genes, and their applications.

[0009] In order to achieve the above object, the first aspect of the present invention provides an Fe(II) / α-ketoglutarate-dependent dioxygenase protein having an amino acid sequence selected from at least one of the following:

[0010] (1) a first amino acid sequence derived from a mutation in at least one of positions 141 and 205 of the amino acid sequence shown in SEQ ID NO: 1;

[0011] (2) a protein derived from the first amino acid sequence in which one or more amino acids are substituted, deleted, or added to the first amino acid sequence, or a protein represented by an amino acid sequence with a tag attached to the amino terminus and / or carboxyl terminus of the first amino acid sequence;

[0012] (3) a second amino acid sequence derived from mutations in at least three positions selected from the group consisting of positions 140, 204, 298, and 335 of the amino acid sequence as shown in SEQ ID NO: 2;

[0013] (4) a protein derived from the second amino acid sequence in which one or more amino acids are substituted, deleted, or added to the second amino acid sequence, or a protein represented by the amino acid sequence with a tag attached to the amino terminus and / or carboxyl terminus of the second amino acid sequence;

[0014] (5) a third amino acid sequence derived from a mutation in at least one of positions selected from positions 206, 301, and 339 of the amino acid sequence as shown in SEQ ID NO: 3;

[0015] (6) a protein derived from the third amino acid sequence in which one or more amino acids are substituted, deleted, or added to the third amino acid sequence, or a protein represented by the amino acid sequence with a tag attached to the amino terminus and / or carboxyl terminus of the third amino acid sequence;

[0016] (7) a fourth amino acid sequence derived from a mutation in at least one of positions selected from positions 142, 207, and 301 of the amino acid sequence shown in SEQ ID NO: 4;

[0017] (8) a protein derived from the fourth amino acid sequence in which one or more amino acids are substituted, deleted, or added to the fourth amino acid sequence, or a protein represented by the amino acid sequence with a tag attached to the amino terminus and / or carboxyl terminus of the fourth amino acid sequence;

[0018] (9) a fifth amino acid sequence derived from a mutation in at least one of positions 285 and 322 of the amino acid sequence as shown in SEQ ID NO: 5;

[0019] (10) a protein derived from the fifth amino acid sequence in which one or more amino acids are substituted, deleted, or added to the fifth amino acid sequence, or a protein represented by the amino acid sequence with a tag attached to the amino terminus and / or carboxyl terminus of the fifth amino acid sequence;

[0020] (11) a sixth amino acid sequence derived from a mutation in at least one of positions selected from positions 142, 207, 301, and 338 of the amino acid sequence of SEQ ID NO: 6;

[0021] (12) a protein derived from the sixth amino acid sequence in which one or more amino acids are substituted, deleted, or added to the sixth amino acid sequence, or a protein represented by the amino acid sequence with a tag attached to the amino terminus and / or carboxyl terminus of the sixth amino acid sequence;

[0022] (13) a seventh amino acid sequence derived from mutations in at least two positions selected from positions 140, 205, and 336 of the amino acid sequence as shown in SEQ ID NO: 7;

[0023] (14) a protein derived from the seventh amino acid sequence in which one or more amino acids are substituted, deleted, or added to the seventh amino acid sequence, or a protein represented by the amino acid sequence with a tag attached to the amino terminus and / or carboxyl terminus of the seventh amino acid sequence;

[0024] (15) an eighth amino acid sequence derived from mutations in at least two positions selected from positions 140, 205, and 336 of the amino acid sequence as shown in SEQ ID NO: 8;

[0025] (16) A protein derived from the eighth amino acid sequence in which one or more amino acids are substituted, deleted, or added to the eighth amino acid sequence, or a protein represented by an amino acid sequence with a tag connected to the amino terminus and / or carboxyl terminus of the eighth amino acid sequence.

[0026] The second aspect of the present invention provides a gene encoding an Fe(II) / α-ketoglutarate-dependent dioxygenase protein, the nucleotide sequence of which is a nucleotide sequence capable of encoding the amino acid sequence of the Fe(II) / α-ketoglutarate-dependent dioxygenase protein described in the first aspect.

[0027] The third aspect of the present invention provides a recombinant vector comprising the gene described in the second aspect.

[0028] The fourth aspect of the present invention provides a transgenic cell comprising the gene according to the second aspect.

[0029] The fifth aspect of the present invention provides a composition comprising the Fe(II) / α-ketoglutarate-dependent dioxygenase protein described in the first aspect.

[0030] The sixth aspect of the present invention provides the use of at least one of the Fe(II) / α-ketoglutarate-dependent dioxygenase protein described in the first aspect, the gene described in the second aspect, the recombinant vector described in the third aspect, and the transgenic cell described in the fourth aspect in improving crop herbicide resistance.

[0031] The seventh aspect of the present invention provides a method for improving crop herbicide resistance, the method comprising: transferring the recombinant vector described in the third aspect into a target plant, so that the target plant expresses the Fe(II) / α-ketoglutarate-dependent dioxygenase protein described in the first aspect to obtain herbicide resistance.

[0032] The eighth aspect of the present invention provides a method for improving crop herbicide resistance, the method comprising: transferring the recombinant vector in a mutant crop containing the recombinant vector described in the third aspect into a target plant through hybridization, breeding or backcrossing, so that the target plant expresses the Fe(II) / α-ketoglutarate-dependent dioxygenase protein described in the first aspect to obtain herbicide resistance.

[0033] The ninth aspect of the present invention provides a method for improving crop herbicide resistance, the method comprising: modifying the Fe(II) / α-ketoglutarate-dependent dioxygenase gene of a target plant by a CRISPR / Cas gene editing method, so that the target plant expresses the Fe(II) / α-ketoglutarate-dependent dioxygenase protein described in the first aspect above to obtain herbicide resistance.

[0034] Compared with the prior art, the Fe(II) / α-ketoglutarate-dependent dioxygenase protein and its encoding gene that are resistant to HPPD inhibitors provided by the present invention have at least the following advantages:

[0035] By introducing the gene encoding the enzyme provided herein into a target plant, the target plant can acquire resistance to HPPD inhibitors (herbicides), while the catalytic activity of the enzyme itself remains substantially unaffected. Therefore, the Fe(II) / α-ketoglutarate-dependent dioxygenase protein and its encoding gene provided herein can be used to cultivate herbicide-resistant plants.

[0036] Other features and advantages of the present invention will be described in detail in the following detailed description. DETAILED DESCRIPTION

[0037] The endpoints of the ranges and any values ​​disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoints of each range, the endpoints of each range and individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered to be specifically disclosed herein.

[0038] As mentioned above, the first aspect of the present invention provides an Fe(II) / α-ketoglutarate-dependent dioxygenase protein having an amino acid sequence selected from at least one of the following:

[0039] (1) a first amino acid sequence derived from a mutation in at least one of positions 141 and 205 of the amino acid sequence shown in SEQ ID NO: 1;

[0040] (2) a protein derived from the first amino acid sequence in which one or more amino acids are substituted, deleted, or added to the first amino acid sequence, or a protein represented by an amino acid sequence with a tag attached to the amino terminus and / or carboxyl terminus of the first amino acid sequence;

[0041] (3) a second amino acid sequence derived from mutations in at least three positions selected from the group consisting of positions 140, 204, 298, and 335 of the amino acid sequence as shown in SEQ ID NO: 2;

[0042] (4) a protein derived from the second amino acid sequence in which one or more amino acids are substituted, deleted, or added to the second amino acid sequence, or a protein represented by the amino acid sequence with a tag attached to the amino terminus and / or carboxyl terminus of the second amino acid sequence;

[0043] (5) a third amino acid sequence derived from a mutation in at least one of positions selected from positions 206, 301, and 339 of the amino acid sequence as shown in SEQ ID NO: 3;

[0044] (6) a protein derived from the third amino acid sequence in which one or more amino acids are substituted, deleted, or added to the third amino acid sequence, or a protein represented by the amino acid sequence with a tag attached to the amino terminus and / or carboxyl terminus of the third amino acid sequence;

[0045] (7) a fourth amino acid sequence derived from a mutation in at least one of positions selected from positions 142, 207, and 301 of the amino acid sequence shown in SEQ ID NO: 4;

[0046] (8) a protein derived from the fourth amino acid sequence in which one or more amino acids are substituted, deleted, or added to the fourth amino acid sequence, or a protein represented by the amino acid sequence with a tag attached to the amino terminus and / or carboxyl terminus of the fourth amino acid sequence;

[0047] (9) a fifth amino acid sequence derived from a mutation in at least one of positions 285 and 322 of the amino acid sequence as shown in SEQ ID NO: 5;

[0048] (10) a protein derived from the fifth amino acid sequence in which one or more amino acids are substituted, deleted, or added to the fifth amino acid sequence, or a protein represented by the amino acid sequence with a tag attached to the amino terminus and / or carboxyl terminus of the fifth amino acid sequence;

[0049] (11) a sixth amino acid sequence derived from a mutation in at least one of positions selected from positions 142, 207, 301, and 338 of the amino acid sequence of SEQ ID NO: 6;

[0050] (12) a protein derived from the sixth amino acid sequence in which one or more amino acids are substituted, deleted, or added to the sixth amino acid sequence, or a protein represented by the amino acid sequence with a tag attached to the amino terminus and / or carboxyl terminus of the sixth amino acid sequence;

[0051] (13) a seventh amino acid sequence derived from mutations in at least two positions selected from positions 140, 205, and 336 of the amino acid sequence as shown in SEQ ID NO: 7;

[0052] (14) a protein derived from the seventh amino acid sequence in which one or more amino acids are substituted, deleted, or added to the seventh amino acid sequence, or a protein represented by the amino acid sequence with a tag attached to the amino terminus and / or carboxyl terminus of the seventh amino acid sequence;

[0053] (15) an eighth amino acid sequence derived from mutations in at least two positions selected from positions 140, 205, and 336 of the amino acid sequence as shown in SEQ ID NO: 8;

[0054] (16) A protein derived from the eighth amino acid sequence in which one or more amino acids are substituted, deleted, or added to the eighth amino acid sequence, or a protein represented by an amino acid sequence with a tag connected to the amino terminus and / or carboxyl terminus of the eighth amino acid sequence.

[0055] In the present invention, unchanged enzyme activity means that under the same assay conditions, the percentage (relative activity) between the enzyme activity of the proteins derived from the first amino acid sequence, the second amino acid sequence, the third amino acid sequence, the fourth amino acid sequence, the fifth amino acid sequence, the sixth amino acid sequence, the seventh amino acid sequence, and the eighth amino acid sequence and the enzyme activity of the wild type is not less than 95% (or 96%, or 97%, or 98%, or 99%, or 100%).

[0056] Preferably, the first amino acid sequence is an amino acid sequence selected from at least one of the following:

[0057] (1a) an amino acid sequence derived from the amino acid sequence shown in SEQ ID NO: 1 after histidine at position 141 is mutated to phenylalanine;

[0058] (1b) an amino acid sequence derived from the amino acid sequence of SEQ ID NO: 1 wherein histidine at position 141 is mutated to phenylalanine and phenylalanine at position 205 is mutated to leucine;

[0059] Preferably, the first amino acid sequence is the amino acid sequence shown by H141F or H141F / F205L.

[0060] According to a specific embodiment of the present invention, the amino acid sequence derived from the mutation of histidine at position 141 in the amino acid sequence shown in SEQ ID NO: 1 to phenylalanine is called H141F.

[0061] According to a specific embodiment of the present invention, the amino acid sequence derived from the mutation of histidine at position 141 to phenylalanine and the mutation of phenylalanine at position 205 to leucine in the amino acid sequence shown in SEQ ID NO: 1 is called H141F / F205L.

[0062] Preferably, the second amino acid sequence is the following amino acid sequence:

[0063] (2a) an amino acid sequence derived from the amino acid sequence of SEQ ID NO: 2 wherein phenylalanine at position 140 is mutated to histidine, leucine at position 204 is mutated to phenylalanine, phenylalanine at position 298 is mutated to leucine, and isoleucine at position 335 is mutated to phenylalanine;

[0064] (2b) an amino acid sequence derived from the amino acid sequence of SEQ ID NO: 2, wherein the leucine at position 204 is mutated to phenylalanine, the phenylalanine at position 298 is mutated to leucine, and the isoleucine at position 335 is mutated to alanine;

[0065] (2c) An amino acid sequence derived from the amino acid sequence shown in SEQ ID NO: 2, wherein the leucine at position 204 is mutated to phenylalanine, the phenylalanine at position 298 is mutated to leucine, and the isoleucine at position 335 is mutated to tryptophan.

[0066] Preferably, the second amino acid sequence is at least one of the amino acid sequences shown by F140H / L204F / F298L / I335F, L204F / F298L / I335A, and L204F / F298L / I335W.

[0067] According to a specific embodiment of the present invention, the amino acid sequence derived from the amino acid sequence shown in SEQ ID NO: 2, in which phenylalanine at position 140 is mutated to histidine, leucine at position 204 is mutated to phenylalanine, phenylalanine at position 298 is mutated to leucine, and isoleucine at position 335 is mutated to phenylalanine, is called F140H / L204F / F298L / I335F.

[0068] According to a specific embodiment of the present invention, the amino acid sequence derived from the amino acid sequence shown in SEQ ID NO: 2, in which the leucine at position 204 is mutated to phenylalanine, the phenylalanine at position 298 is mutated to leucine, and the isoleucine at position 335 is mutated to alanine, is called L204F / F298L / I335A.

[0069] According to a specific embodiment of the present invention, the amino acid sequence derived from the amino acid sequence shown in SEQ ID NO: 2 in which the leucine at position 204 is mutated to phenylalanine, the phenylalanine at position 298 is mutated to leucine, and the isoleucine at position 335 is mutated to tryptophan is called L204F / F298L / I335W.

[0070] Preferably, the third amino acid sequence is the following amino acid sequence:

[0071] (3a) an amino acid sequence derived from the amino acid sequence of SEQ ID NO: 3 wherein the threonine at position 206 is mutated to phenylalanine;

[0072] (3b) an amino acid sequence derived from the amino acid sequence of SEQ ID NO: 3 wherein the threonine at position 206 is mutated to phenylalanine and the phenylalanine at position 301 is mutated to leucine;

[0073] (3c) An amino acid sequence derived from the amino acid sequence shown in SEQ ID NO: 3, wherein the threonine at position 206 is mutated to phenylalanine, the phenylalanine at position 301 is mutated to leucine, and the tyrosine at position 339 is mutated to phenylalanine.

[0074] Preferably, the third amino acid sequence is at least one of the amino acid sequences shown by T206F, T206F / F301L, and T206F / F301L / Y339F.

[0075] According to a specific embodiment of the present invention, the amino acid sequence derived from the mutation of threonine at position 206 in the amino acid sequence shown in SEQ ID NO: 3 to phenylalanine is called T206F.

[0076] According to a specific embodiment of the present invention, the amino acid sequence derived from the mutation of threonine at position 206 to phenylalanine and the mutation of phenylalanine at position 301 to leucine in the amino acid sequence shown in SEQ ID NO: 3 is called T206F / F301L.

[0077] According to a specific embodiment of the present invention, the amino acid sequence derived from the mutation of threonine at position 206 to phenylalanine, the mutation of phenylalanine at position 301 to leucine, and the mutation of tyrosine at position 339 to phenylalanine in the amino acid sequence shown in SEQ ID NO: 3 is called T206F / F301L / Y339F.

[0078] Preferably, the fourth amino acid sequence is the following amino acid sequence:

[0079] (4a) an amino acid sequence derived from the amino acid sequence of SEQ ID NO: 4 wherein tyrosine at position 142 is mutated to histidine;

[0080] (4b) an amino acid sequence derived from the amino acid sequence of SEQ ID NO: 4 in which tyrosine at position 142 is mutated to histidine and isoleucine at position 207 is mutated to phenylalanine;

[0081] (4c) An amino acid sequence derived from the amino acid sequence shown in SEQ ID NO: 4, wherein tyrosine at position 142 is mutated to histidine, isoleucine at position 207 is mutated to phenylalanine, and phenylalanine at position 301 is mutated to leucine.

[0082] Preferably, the fourth amino acid sequence is at least one of the amino acid sequences shown by Y142H, Y142H / I207F, and Y142H / I207F / F301L.

[0083] According to a specific embodiment of the present invention, the amino acid sequence derived from the mutation of tyrosine at position 142 in the amino acid sequence shown in SEQ ID NO: 4 to histidine is called Y142H.

[0084] According to a specific embodiment of the present invention, the amino acid sequence derived from the mutation of tyrosine at position 142 to histidine and the mutation of isoleucine at position 207 to phenylalanine in the amino acid sequence shown in SEQ ID NO: 4 is called Y142H / I207F.

[0085] According to a specific embodiment of the present invention, the amino acid sequence derived from the mutation of tyrosine at position 142 to histidine, the mutation of isoleucine at position 207 to phenylalanine, and the mutation of phenylalanine at position 301 to leucine in the amino acid sequence shown in SEQ ID NO: 4 is called Y142H / I207F / F301L.

[0086] Preferably, the fifth amino acid sequence is the following amino acid sequence:

[0087] (5a) an amino acid sequence derived from the amino acid sequence of SEQ ID NO: 5 wherein the phenylalanine at position 285 is mutated to leucine;

[0088] (5b) An amino acid sequence derived from the amino acid sequence shown in SEQ ID NO: 5 in which the phenylalanine at position 285 is mutated to leucine and the histidine at position 322 is mutated to phenylalanine.

[0089] Preferably, the fifth amino acid sequence is at least one of the amino acid sequences shown by F285L and F285L / H322F.

[0090] According to a specific embodiment of the present invention, the amino acid sequence derived from the mutation of phenylalanine at position 285 in the amino acid sequence shown in SEQ ID NO: 5 to leucine is called F285L.

[0091] According to a specific embodiment of the present invention, the amino acid sequence derived from the mutation of phenylalanine at position 285 to leucine and the mutation of histidine at position 322 to phenylalanine in the amino acid sequence shown in SEQ ID NO: 5 is called F285L / H322F.

[0092] Preferably, the sixth amino acid sequence is the following amino acid sequence:

[0093] (6a) an amino acid sequence derived from the amino acid sequence of SEQ ID NO: 6 wherein glutamine at position 142 is mutated to histidine;

[0094] (6b) an amino acid sequence derived from the amino acid sequence of SEQ ID NO: 6 wherein glutamine at position 142 is mutated to histidine and isoleucine at position 207 is mutated to phenylalanine;

[0095] (6c) an amino acid sequence derived from the amino acid sequence of SEQ ID NO: 6 wherein glutamine at position 142 is mutated to histidine, isoleucine at position 207 is mutated to phenylalanine, and phenylalanine at position 301 is mutated to leucine;

[0096] (6d) an amino acid sequence derived from the amino acid sequence of SEQ ID NO: 6 in which glutamine at position 142 is mutated to histidine, isoleucine at position 207 is mutated to phenylalanine, phenylalanine at position 301 is mutated to leucine, and arginine at position 338 is mutated to phenylalanine;

[0097] (6e) An amino acid sequence derived from the amino acid sequence shown in SEQ ID NO: 6 wherein glutamine at position 142 is mutated to histidine and arginine at position 338 is mutated to phenylalanine.

[0098] Preferably, the sixth amino acid sequence is at least one of the amino acid sequences shown by Q142H, Q142H / I207F, Q142H / I207F / F301L, Q142H / I207F / F301L / R338F, and Q142H / R338F.

[0099] According to a specific embodiment of the present invention, the amino acid sequence derived from the mutation of glutamine at position 142 in the amino acid sequence shown in SEQ ID NO: 6 to histidine is called Q142H.

[0100] According to a specific embodiment of the present invention, the amino acid sequence derived from the mutation of glutamine at position 142 to histidine and the mutation of isoleucine at position 207 to phenylalanine in the amino acid sequence shown in SEQ ID NO: 6 is called Q142H / I207F.

[0101] According to a specific embodiment of the present invention, the amino acid sequence derived from the amino acid sequence shown in SEQ ID NO: 6 in which glutamine at position 142 is mutated to histidine, isoleucine at position 207 is mutated to phenylalanine, and phenylalanine at position 301 is mutated to leucine is called Q142H / I207F / F301L.

[0102] According to a specific embodiment of the present invention, the amino acid sequence derived from the amino acid sequence shown in SEQ ID NO:6 in which glutamine at position 142 is mutated to histidine, isoleucine at position 207 is mutated to phenylalanine, phenylalanine at position 301 is mutated to leucine, and arginine at position 338 is mutated to phenylalanine is called Q142H / I207F / F301L / R338F.

[0103] According to a specific embodiment of the present invention, the amino acid sequence derived from the mutation of glutamine at position 142 to histidine and the mutation of arginine at position 338 to phenylalanine in the amino acid sequence shown in SEQ ID NO: 6 is called Q142H / R338F.

[0104] Preferably, the seventh amino acid sequence is the following amino acid sequence:

[0105] (7a) an amino acid sequence derived from the amino acid sequence of SEQ ID NO: 7 in which glutamine at position 140 is mutated to histidine and tyrosine at position 205 is mutated to phenylalanine;

[0106] (7b) An amino acid sequence derived from the amino acid sequence shown in SEQ ID NO: 7 in which glutamine at position 140 is mutated to histidine, tyrosine at position 205 is mutated to phenylalanine, and lysine at position 336 is mutated to phenylalanine.

[0107] Preferably, the seventh amino acid sequence is at least one of the amino acid sequences shown by Q140H / Y205F and Q140H / Y205F / K336F.

[0108] According to a specific embodiment of the present invention, the amino acid sequence derived from the mutation of glutamine at position 140 to histidine and the mutation of tyrosine at position 205 to phenylalanine in the amino acid sequence shown in SEQ ID NO: 7 is called Q140H / Y205F sequence.

[0109] According to a specific embodiment of the present invention, the amino acid sequence derived from the amino acid sequence shown in SEQ ID NO: 7 in which glutamine at position 140 is mutated to histidine, tyrosine at position 205 is mutated to phenylalanine, and lysine at position 336 is mutated to phenylalanine is called Q140H / Y205F / K336F sequence.

[0110] Preferably, the eighth amino acid sequence is the following amino acid sequence:

[0111] (8a) an amino acid sequence derived from the amino acid sequence of SEQ ID NO: 8 in which glutamine at position 140 is mutated to histidine and tyrosine at position 205 is mutated to phenylalanine;

[0112] (8b) An amino acid sequence derived from the amino acid sequence shown in SEQ ID NO: 8 in which glutamine at position 140 is mutated to histidine, tyrosine at position 205 is mutated to phenylalanine, and lysine at position 336 is mutated to phenylalanine.

[0113] Preferably, the eighth amino acid sequence is at least one of the amino acid sequences shown by Q140H / Y205F and Q140H / Y205F / K336F.

[0114] According to a specific embodiment of the present invention, the amino acid sequence derived from the mutation of glutamine at position 140 to histidine and the mutation of tyrosine at position 205 to phenylalanine in the amino acid sequence shown in SEQ ID NO: 8 is called Q140H / Y205F.

[0115] According to a specific embodiment of the present invention, the amino acid sequence derived from the amino acid sequence shown in SEQ ID NO: 8 in which glutamine at position 140 is mutated to histidine, tyrosine at position 205 is mutated to phenylalanine, and lysine at position 336 is mutated to phenylalanine is called Q140H / Y205F / K336F.

[0116] In the present invention, methods for obtaining the above-mentioned proteins are well known to those skilled in the art. For example, when the amino acid sequence of the protein is known, the protein can be obtained directly by chemical synthesis, or by first obtaining the gene encoding the protein and then obtaining the protein by biological expression.

[0117] The proteins provided herein may also be modified. Modifications (generally without altering the primary structure, i.e., without altering the amino acid sequence) include chemical derivatization of proteins in vivo or in vitro, such as acetylation or hydroxylation. Modifications also include glycosylation, such as those resulting from glycosylation during protein synthesis and processing or in further processing steps, which can be accomplished by exposing the protein to glycosylation enzymes (e.g., mammalian glycosylases or deglycosylases). Modifications also include sequences containing phosphorylated amino acid residues (e.g., phosphotyrosine, phosphoserine, phosphothreonine).

[0118] In the present invention, to facilitate purification, proteins can also be modified using tags commonly used in the art. For example, this can be achieved by attaching a common purification tag (e.g., at least one of GST, Poly-His, FLAG, His-SUMO, and c-myc) listed in Table 1 to the amino and / or carboxyl termini of the protein. Such tags will not affect the activity of the proteins provided herein. In actual applications, whether to add tags can be selected based on needs.

[0119] Table 1

[0120] As described above, the second aspect of the present invention provides a gene encoding an Fe(II) / α-ketoglutarate-dependent dioxygenase protein, the nucleotide sequence of which is a nucleotide sequence capable of encoding the amino acid sequence of the Fe(II) / α-ketoglutarate-dependent dioxygenase protein described in the first aspect.

[0121] Preferably, the nucleotide sequence of the gene is at least one nucleotide sequence selected from the group consisting of:

[0122] (1) the nucleotide sequence shown in SEQ ID NO: 11;

[0123] (2) a nucleotide sequence that is at least 90% identical, preferably at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 11;

[0124] (3) the nucleotide sequence shown in SEQ ID NO: 21;

[0125] (4) a nucleotide sequence that is at least 90% identical, preferably at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 21;

[0126] (5) the nucleotide sequence shown in SEQ ID NO: 31;

[0127] (6) a nucleotide sequence that is at least 90% identical, preferably at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 31;

[0128] (7) the nucleotide sequence shown in SEQ ID NO:41;

[0129] (8) a nucleotide sequence that is at least 90% identical, preferably at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 41;

[0130] (9) the nucleotide sequence shown in SEQ ID NO:51;

[0131] (10) a nucleotide sequence that is at least 90% identical, preferably at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 51;

[0132] (11) the nucleotide sequence shown in SEQ ID NO: 61;

[0133] (12) a nucleotide sequence that is at least 90% identical, preferably at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 61;

[0134] (13) the nucleotide sequence shown in SEQ ID NO:71;

[0135] (14) a nucleotide sequence that is at least 90% identical, preferably at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 71;

[0136] (15) the nucleotide sequence shown in SEQ ID NO: 81;

[0137] (16) A nucleotide sequence that is at least 90% identical, preferably at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO:81.

[0138] In the present invention, it is well known in the art that, of the 20 different amino acids that make up proteins, with the exception of Met (ATG) and Trp (TGG), which are each encoded by a single codon, the other 18 amino acids are encoded by 2-6 codons (Sambrook et al., Molecular Cloning, Cold Spring Harbor Laboratory Press, New York, USA, 2nd edition, 1989, see Appendix D on page 950). That is, due to the degeneracy of the genetic code, there is often more than one codon that specifies an amino acid. Substitution of the third nucleotide in a triplet codon generally does not change the amino acid composition, and therefore, the nucleotide sequences of genes encoding the same protein can differ.

[0139] Those skilled in the art can fully deduce the nucleotide sequences of the genes encoding the amino acid sequences SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 and the specific mutation patterns of the proteins provided in the present invention based on the known codon table, and obtain the nucleotide sequences by biological methods (such as PCR methods, mutation methods) or chemical synthesis methods. Therefore, this partial nucleotide sequence should all be included in the scope of the present invention.

[0140] Similarly, using the nucleotide sequences SEQ ID NO: 11, SEQ ID NO: 21, SEQ ID NO: 31, SEQ ID NO: 41, SEQ ID NO: 51, SEQ ID NO: 61, SEQ ID NO: 71, SEQ ID NO: 81 and the specific mutation methods of the proteins provided by the present invention, the amino acid sequences provided by the present invention can also be obtained by modifying SEQ ID NO: 11, SEQ ID NO: 21, SEQ ID NO: 31, SEQ ID NO: 41, SEQ ID NO: 51, SEQ ID NO: 61, SEQ ID NO: 71, SEQ ID NO: 81 by methods well known in the art, such as the method of Sambrook et al. (Molecular Cloning, Cold Spring Harbor Laboratory Press, New York, USA, second edition, 1989).

[0141] In the present invention, based on the above-mentioned protein and nucleotide sequences SEQ ID NO: 11, SEQ ID NO: 21, SEQ ID NO: 31, SEQ ID NO: 41, SEQ ID NO: 51, SEQ ID NO: 61, SEQ ID NO: 71, and SEQ ID NO: 81, a person skilled in the art can easily obtain the gene encoding the protein provided by the present invention. For example, site-directed mutagenesis can be performed on the basis of SEQ ID NO: 11, and the site-directed mutagenesis method includes but is not limited to ZFN site-directed mutagenesis method, TALEN site-directed mutagenesis method, and / or genomic site-directed mutagenesis method such as CRISPR-Cas9.

[0142] In the present invention, as described in the first aspect, correspondingly, the 5' end and / or 3' end of the nucleotide sequence provided by the present invention may further be connected to the coding sequence of the common purification tags shown in Table 1.

[0143] The nucleotide sequence provided by the present invention can usually be obtained by polymerase chain reaction (PCR) amplification, recombinant method, or artificial synthesis method. Illustratively, those skilled in the art can easily obtain templates and primers based on the nucleotide sequence provided by the present invention, and utilize PCR to amplify and obtain the nucleotide sequence. After obtaining the nucleotide sequence, the amino acid sequence can be obtained in large quantities by recombinant method. Usually the obtained nucleotide sequence is cloned into a vector, then transferred into genetically engineered bacteria, and then the nucleotide sequence is obtained by conventional method from the host cell separation after propagation. In addition, the nucleotide sequence can also be synthesized by artificial chemical synthesis methods well known in the art.

[0144] As mentioned above, the third aspect of the present invention provides a recombinant vector containing the gene described in the second aspect.

[0145] In the present invention, the "vector" used in the recombinant vector can be selected from various vectors known in the art, such as various commercially available plasmids, cosmids, phages, and retroviruses, and can be selected according to specific circumstances. For example, it can be pGWC, pB2GW7.0, or pET-28a. Recombinant vector construction can be performed by enzyme digestion with various nucleases that have cleavage sites in the vector's multiple cloning site to obtain a linearized plasmid, which can then be ligated with a gene fragment cleaved with the same nuclease to obtain a recombinant plasmid.

[0146] As mentioned above, the fourth aspect of the present invention provides a transgenic cell, which contains the gene described in the second aspect.

[0147] Preferably, the transgenic cell is a prokaryotic cell.

[0148] In the present invention, the recombinant vector can be transformed, transduced, or transfected into a host cell by methods known in the art, such as chemical transformation using the calcium chloride method or high-voltage electroporation, preferably electroporation. The host cell can be a prokaryotic cell or a eukaryotic cell, and the selection can be based on the actual situation. The cell can be a DH5α strain, Agrobacterium tumefaciens strain GV3101, or the like.

[0149] As mentioned above, the fifth aspect of the present invention provides a composition comprising the Fe(II) / α-ketoglutarate-dependent dioxygenase protein described in the first aspect.

[0150] The composition provided by the present invention contains the Fe(II) / α-ketoglutarate-dependent dioxygenase protein described in the first aspect of the present invention as an active ingredient, with the protein content being 50-90% by weight, based on the total weight of the composition. The composition may further contain a solvent known to those skilled in the art (e.g., glycerol, sugars, and protein protectants such as protease inhibitors).

[0151] As described above, the sixth aspect of the present invention provides the use of at least one of the Fe(II) / α-ketoglutarate-dependent dioxygenase protein described in the first aspect, the gene described in the second aspect, the recombinant vector described in the third aspect, and the transgenic cell described in the fourth aspect in improving crop herbicide resistance.

[0152] Preferably, the herbicide is an HPPD inhibitor.

[0153] Preferably, the herbicide is at least one of a triketone compound, a pyrazole compound, an isoxazole compound, a diketonitrile compound and a heterocyclic amide compound.

[0154] More preferably, the herbicide is at least one of mesotrione, tampoxetone, furazolidinone, bicyclonazole, quinclorac (Y13161), methylquinclorac (Y13287), Y18024, Y16550 and pyraclostrobin, and its molecular structure is as follows:

[0155] As mentioned above, the seventh aspect of the present invention provides a method for improving crop herbicide resistance, the method comprising: transferring the recombinant vector described in the third aspect into a target plant, so that the target plant expresses the Fe(II) / α-ketoglutarate-dependent dioxygenase protein described in the first aspect to obtain resistance to the herbicide.

[0156] As described above, the eighth aspect of the present invention provides a method for improving crop herbicide resistance, the method comprising: transferring the recombinant vector in the mutant crop containing the recombinant vector described in the third aspect into the target plant through hybridization, breeding or backcrossing, so that the target plant expresses the Fe(II) / α-ketoglutarate-dependent dioxygenase protein described in the first aspect to obtain resistance to the herbicide.

[0157] As mentioned above, the ninth aspect of the present invention provides a method for improving crop herbicide resistance, the method comprising: modifying the HIS1 / HSL gene of the target plant by the CRISPR / Cas gene editing method, so that the target plant expresses the Fe(II) / α-ketoglutarate-dependent dioxygenase protein described in the first aspect above to obtain resistance to the herbicide.

[0158] In the present invention, introducing the recombinant vector into the target plant specifically refers to: introducing the nucleotide sequence of the herbicide-resistant protein provided by the present invention into the target plant through genetic engineering methods, thereby conferring resistance to HPPD inhibitor herbicides on the target plant. Alternatively, the nucleotide sequence of the protein provided by the present invention can be introduced into the target plant through hybridization, breeding, backcrossing, and other methods to confer resistance to HPPD inhibitor herbicides on the target plant. Furthermore, the HIS1 / HSL gene of the target plant can be directly modified using gene editing technologies such as CRISPR / Cas to confer resistance to HPPD inhibitor herbicides on the target plant. More specifically, the protein can be used as a parental material to be hybridized with other superior plant varieties and further backcrossed to further transfer the herbicide-resistant trait to other target plant varieties.

[0159] The transgenic methods used in the present invention are well known to those skilled in the art, and include direct or indirect transformation methods. Direct transformation methods include chemical induction, liposome method, gene gun method, electroporation method, and microinjection method.

[0160] In the present invention, the term "plant" has the broadest meaning. Examples of plants include, but are not limited to, vascular plants, vegetables, grains, flowers, trees, herbs, shrubs, grasses, vines, ferns, mosses, fungi and algae, as well as clones and plant parts used for asexual reproduction (e.g., cuttings, layering, shoots, rhizomes, underground stems, culms, root collars, bulbs, corms, tubers, rhizomes, plants / tissues produced in tissue culture, etc.). The term "plant" further encompasses whole plants, plant parents and offspring, and plant parts, including seed, branch, stem, leaf, root (including tuber), flower, floret, fruit, fleshy stem, peduncle, stamen, anther, stigma, style, ovary, petal, sepal, carpel, root tip, root cap, root hair, leaf hair, seed hair, pollen grain, microspore, cotyledon, hypocotyl, epicotyl, xylem, phloem, parenchyma, endosperm, companion cell, guard cell, and any other known organ, tissue and cell, and tissue and organ of plant. The term "plant" also encompasses plant cells, suspension cultures, callus, embryo, meristematic zone, gametophyte, sporophyte, pollen and microspore. Wherein, the above-mentioned all encompasses target gene / nucleic acid provided by the invention.

[0161] Plants particularly suitable for use in the methods of the invention include all plants belonging to the superfamily Viridiplantae, in particular monocotyledonous and dicotyledonous plants, including food crops, fodder or forage legumes, ornamental plants, trees or shrubs.

[0162] According to a preferred embodiment of the present invention, the plant is a crop plant. Examples of crop plants include rice, wheat, corn, sorghum, soybean, sunflower, rape, alfalfa, cotton, tomato, potato or tobacco. More preferably, the crop plant is a cereal, such as rice, wheat, corn, sorghum, barley, millet, rye or oats.

[0163] The beneficial effects achieved by the present invention are obtaining proteins, genes, recombinant vectors, transgenic cells, compositions, and plants that can replace existing technologies for making crops resistant to herbicides, and applications and methods for obtaining crops with herbicide resistance. In addition, crop varieties with herbicide resistance can be obtained through transgenic or non-transgenic methods, and weed control in cultivated paddy fields and cultivated dry fields can be carried out more efficiently, thereby increasing crop yield and yield stability.

[0164] In the following examples, unless otherwise specified, room temperature refers to 25±2°C.

[0165] In the following examples, unless otherwise specified, the experimental methods used are conventional methods.

[0166] In the following examples, unless otherwise specified, the materials and reagents involved can be obtained from commercial sources.

[0167] Agarose gel recovery kit was purchased from TIANGEN;

[0168] Restriction enzymes and supporting solutions were purchased from Takara;

[0169] The ligation kit was purchased from New England BioLabs.

[0170] Example 1

[0171] Synthesis of coding genes and nucleotide sequences

[0172] The following coding genes and nucleotide sequences were synthesized by Wuhan Jinkairui Bioengineering Co., Ltd. based on the nucleotide sequence provided by the present invention.

[0173] (1) SEQ ID NO: 11

[0174] According to the nucleotide sequence shown in SEQ ID NO: 11, the coding gene of the protein shown in Table 2 and the nucleotide sequence shown in SEQ ID NO: 11 were synthesized.

[0175] Table 2

[0176] (2) SEQ ID NO: 21

[0177] Based on the nucleotide sequence shown in SEQ ID NO: 21, the coding gene for the protein shown in Table 3 and the nucleotide sequence shown in SEQ ID NO: 21 were synthesized.

[0178] Table 3

[0179] (3) SEQ ID NO: 31

[0180] Based on the nucleotide sequence shown in SEQ ID NO: 31, the gene encoding the protein shown in Table 4 and the nucleotide sequence shown in SEQ ID NO: 31 were synthesized.

[0181] Table 4

[0182] (4) SEQ ID NO: 41

[0183] Based on the nucleotide sequence shown in SEQ ID NO: 41, the gene encoding the protein shown in Table 5 and the nucleotide sequence shown in SEQ ID NO: 41 were synthesized.

[0184] Table 5

[0185] (5) SEQ ID NO: 51

[0186] Based on the nucleotide sequence shown in SEQ ID NO: 51, the coding gene for the protein shown in Table 6 and the nucleotide sequence shown in SEQ ID NO: 51 were synthesized.

[0187] Table 6

[0188] (6) SEQ ID NO: 61

[0189] Based on the nucleotide sequence shown in SEQ ID NO: 61, the coding gene for the protein shown in Table 7 and the nucleotide sequence shown in SEQ ID NO: 61 were synthesized.

[0190] Table 7

[0191] (7) SEQ ID NO: 71

[0192] Based on the nucleotide sequence shown in SEQ ID NO: 71, the gene encoding the protein shown in Table 8 and the nucleotide sequence shown in SEQ ID NO: 71 were synthesized.

[0193] Table 8

[0194] (8) SEQ ID NO: 81

[0195] Based on the nucleotide sequence shown in SEQ ID NO: 81, the gene encoding the protein shown in Table 9 and the nucleotide sequence shown in SEQ ID NO: 81 were synthesized.

[0196] Table 9

[0197] Example 2

[0198] Construction of expression vector

[0199] (1) Amplification of target gene

[0200] Using the nucleotide sequences of SEQ ID NO: 11, SEQ ID NO: 21, SEQ ID NO: 31, SEQ ID NO: 41, SEQ ID NO: 51, SEQ ID NO: 61, SEQ ID NO: 71, and SEQ ID NO: 81 as templates, PCR reactions were performed to obtain the wild-type and mutant gene sequences of HIS1 / HSL shown in Tables 2 to 9, and ligated into the corresponding vectors.

[0201] The PCR reaction system and PCR program settings are shown in Table 10 and Table 11 respectively:

[0202] Table 10

[0203] Table 11

[0204] The extension time at 72°C depends on the specific fragment length and the efficiency of the enzyme used. Steps 2 to 4 in the above procedure were repeated for 25 cycles.

[0205] (2) Agarose gel electrophoresis of nucleic acids and gel recovery of PCR products

[0206] a. Weigh 1g of agarose into a conical flask and add 100mL of 1×TAE solution (to make a 50x stock solution, prepare as follows: weigh 242g of Tris and 18.6g of EDTA into a 1L beaker; add approximately 800mL of deionized water and stir; add 57.1mL of glacial acetic acid and dissolve thoroughly; adjust the pH to 8.3 with NaOH, then add deionized water to 1L and store at room temperature. Heat in a microwave oven until the agarose particles are completely dissolved. After cooling until cool to the touch, add 5μL of ethidium bromide solution, mix thoroughly, and pour into a gel casting tank with a comb in place. Allow to cool and solidify.

[0207] b. Remove the comb immediately before use, place the gel in the electrophoresis tank, and add an appropriate amount of 1×TAE solution. Add the sample to the well, close the lid, and start electrophoresis. Stop electrophoresis when the bromophenol blue moves to 1 / 3 of the distance from the bottom.

[0208] c. Excise the target band, place it into a clean 1.5 mL EP tube, and proceed to gel recovery. The gel recovery kit was purchased from TIANGEN (Cat. No. DP209). For every 100 mg of gel, add 300 μL of gel solution PN (provided with the kit). Incubate the tube in a 50°C water bath, mixing by inverting the tube to dissolve the gel.

[0209] d. Add the solution in c to the pretreated adsorption column (provided in the kit), let it stand at room temperature for 5 minutes to allow it to fully bind, and then centrifuge at 12000 rpm for 1 minute;

[0210] e. Pour out the liquid in the collection tube and repeat step d once to increase the recovery rate of the product;

[0211] f. Add 500 μL of wash solution PW (provided in the kit) to the purification column and centrifuge at 12,000 rpm for 1 min; repeat this operation once;

[0212] g. Centrifuge the adsorption column at 13000 rpm for 2 min to remove the PW solution as much as possible;

[0213] h. Place the purification column in a new EP tube and heat it at 65°C for 10 minutes to evaporate the PW solution.

[0214] i. Add 50 μL of elution solution (provided with the kit) dropwise to the middle of the adsorption column, place on a 65°C heating table for 2 minutes, and centrifuge at 13,000 rpm for 2 minutes to elute the DNA; repeat this step once;

[0215] j. Finally, store the recovered PCR products at -20°C.

[0216] (3) Enzyme digestion reaction

[0217] After electrophoresis verification of the correct size of DNA fragments, the vector and PCR product are double-digested with enzymes (the enzyme digestion solution is purchased together with the restriction endonuclease). The enzyme digestion system is shown in Table 12:

[0218] Table 12

[0219] After incubation at 37°C for 3 h, electrophoresis was performed and the fragments were recovered using a gel recovery kit. To improve the efficiency of vector cleavage, 1.5 μL of restriction endonuclease was added and the cleavage time was extended by 5 h.

[0220] (4) Ligation reaction

[0221] The recovered gene fragments were ligated using the following system. The ligation reagents, Solution 1 (containing T4 DNA ligase and supporting buffer), were purchased from New England BioLabs (Cat. #M0202S). The fragments were then incubated at 16°C for at least 5 hours. The ligation system is shown in Table 13:

[0222] Table 13

[0223] (5) Conversion

[0224] After the ligation is completed, the ligation product is transformed into Escherichia coli JM109 competent cells (purchased from Promega, product number ST1105) and cultured. The specific steps are as follows:

[0225] a. Take out a tube of competent cells and place it on ice to thaw for 10 minutes;

[0226] b. Add the ligation product to the competent cells, mix well, and let it stand on ice for 30 minutes;

[0227] c. Heat shock at 42°C for 90 seconds, then place on ice for 2 minutes;

[0228] d. Add 200 μL of Luria-Bertani (LB) medium (without antibiotics) to the tube and incubate at 37°C, 220 rpm for 30 min; preheat the solid LB plate with the corresponding resistance to room temperature;

[0229] e. Take out the bacterial solution and spread it evenly on the plate, and culture it in a 37℃ incubator for 16 hours.

[0230] (6) Sequencing

[0231] Select some single clones from the plate cultured for 16 hours and transfer them to a clean plate. Incubate them at 37°C for 5 hours and then send them to Wuhan Jinkairui Bioengineering Co., Ltd. for sequencing. After the sequencing results are fed back, sequence comparison is performed to confirm the correct clone.

[0232] Example 3

[0233] Expression and purification of target proteins

[0234] (1) Expression

[0235] First, perform a small protein extraction to explore the optimal expression conditions and purification strategy. Once the conditions are determined, perform a large-scale extraction. Protein expression is performed in Escherichia coli BL21 (DE3) cells (purchased from Kangti Life Science Technology Co., Ltd., product number KTSM109) using the following process:

[0236] a. Pick a single colony from the plate after 16 hours of culture and add it to 100 mL of LB medium containing the desired resistance antibiotic. Incubate at 37°C and 220 rpm for 5 hours until noticeable turbidity is observed.

[0237] b. Expand the vial of bacteria from step a into a large bottle of LB medium (containing antibiotics) at a volume ratio of 1:100 and culture at 37°C and 220 rpm for 3 h. After the OD600 value reaches 0.7 h, reduce the temperature to 20°C and add 0.2 mM isopropyl-β-D-thiogalactopyranoside (IPTG) for induction for 14 h.

[0238] c. Centrifuge at 4°C, 4000 rpm for 10 min, collect the bacteria, and use them for protein purification.

[0239] (2) Purification

[0240] a. Resuspend the collected E. coli cells in a cell lysis solution (30 mL:1 L LB culture medium). Stir on a magnetic stirrer for 20 minutes to mix the cells. During this time, add 1 mM of the serine protease inhibitor phenylmethylsulfonyl fluoride (PMSF), 40 μg / mL of lysozyme to lyse bacterial cell walls, 1 μg / mL of deoxyribonuclease I to degrade nucleic acids, and 10 mM of the cofactor MgCl2. The cells are then ultrasonically disrupted.

[0241] b. After sonication, centrifuge at 13,000 rpm and 4°C for 1 hour, and collect the supernatant for affinity chromatography; protein purification was performed in a constant temperature chamber at 4°C;

[0242] c. First, the supernatant after centrifugation was poured into the treated Ni column for full binding; then, it was rinsed with a solution containing different concentrations (10mM, 30mM, 50mM) of imidazole to remove impurities; finally, the target protein was eluted with a solution containing a high concentration (250mM) of imidazole; and the results were analyzed by SDS-PAGE;

[0243] d. The protein after affinity chromatography was diluted and loaded onto an ion exchange column. After loading, it was eluted using a linear gradient (NaCl from 0 to 500 mM) with a NaCl buffer solution (Tris buffer, pH 7.0) using a protein purifier. The results were analyzed by SDS-PAGE, and proteins with good properties and purity were pooled and concentrated for the next step of molecular sieve chromatography.

[0244] e. Before loading the sample onto the molecular sieve chromatography column, centrifuge at 13,000 rpm for 5 minutes to remove some precipitated protein and impurities. Then, transfer the sample to the loading loop using a syringe. Elute the protein with elution buffer (containing 100 mM NaCl, Tris, pH 7.0), collecting 0.5 mL of sample per tube. Analyze the protein using SDS-PAGE. Pool the proteins to be stored, then aliquot and freeze. Proteins to be used for activity testing should be stored in glycerol at a final concentration of 30% by volume.

[0245] Test Example 1

[0246] Study on the catalytic activity of HPPD inhibitors

[0247] Under the premise of ensuring that the concentrations of each protein and HPPD inhibitor are the same, high performance liquid chromatography (HPLC) is used to test the catalytic rate and degree of each protein on the HPPD inhibitor.

[0248] The specific test method steps are as follows:

[0249] a. Preparation of buffer solution, substrate and cofactor:

[0250] HEPES buffer for activity measurement: Prepare a 1 M stock solution, adjust its pH to 7.0 with sodium hydroxide, dilute to 20 mM immediately before use, and filter through a 0.22 μm filter.

[0251] Preparation of substrate (i.e., HPPD inhibitor): Prepare a 10 mM stock solution in DMSO and dilute with activity assay buffer just before use;

[0252] Preparation of sodium ascorbate: Prepare to a concentration of 20 mM with deionized water. Store at -80°C.

[0253] Preparation of ferrous sulfate: Prepare to a concentration of 1 mM with deionized water. Store at -80°C.

[0254] Preparation of α-ketoglutarate: Prepare to a concentration of 50 mM with deionized water. Store at -80°C.

[0255] b. For the assay, first mix the activity buffer (final concentration 20 mM), substrate (final concentration 50 μM), sodium ascorbate (final concentration 2 mM), ferrous sulfate (final concentration 100 μM), and α-ketoglutarate (final concentration 1 mM). Finally, add the enzyme (final concentration 0.5 mg / mL) to a total volume of 200 μL and incubate at 30°C for 10 h.

[0256] c. Add 20 μL of 10% trifluoroacetic acid by volume to terminate the reaction, centrifuge at 12000 rpm for 10 min, and take 180 μL into a sample vial for testing;

[0257] d. When testing samples by HPLC, load 60 μL of sample and measure the residual substrate absorption peak area at 286 nm. Calculate the amount of substrate consumed in different reactions based on a standard curve (i.e., control) to evaluate the catalytic activity of each protein. Repeat three times for each sample.

[0258] e. Determination of substrate (HPPD inhibitor) standard curve: Using the same reaction system as above (but without enzyme), different substrate concentrations were set, such as 0.1 μM, 0.5 μM, 1 μM, 10 μM, and 50 μM, to obtain the relationship between substrate concentration and peak area.

[0259] Control: refers to adding inhibitors to the reaction system but not adding enzymes, which is regarded as the remaining amount of substrate when the inhibitor is not catalyzed.

[0260] Sample: Add the same volume and concentration (50 μM) of inhibitor to the enzyme reaction system and observe the catalytic efficiency of the enzyme on the inhibitor.

[0261] Catalytic efficiency = (1-(peak area of ​​substrate in sample) / (peak area of ​​substrate in control)) × 100%. The larger the value, the better the catalytic effect.

[0262] This test case tested two types of representative HPPD inhibitor herbicides:

[0263] Triketones: mesotrione, cypermethrin, furazolidinone, bicyclonazole, methylquinazoline, and compound A;

[0264] Pyrazoles: pyraclostrobin technical and pyraclostrobin (Baowei), the catalytic results of rice HIS1 protein and HSL proteins of rice, wheat, barley, sorghum and corn on the above HPPD inhibitors were tested respectively; the specific results are shown in Tables 14 to 21.

[0265] Table 14: Catalytic results of rice HIS1 mutants towards different HPPD inhibitors

[0266] In Table 14, H141F refers to a rice HIS1 mutant in which the 141st position of the histidine was mutated to the phenylalanine; H141F / F205L refers to a rice HIS1 mutant in which the 141st position of the histidine was mutated to the phenylalanine and the 205th position of the phenylalanine was mutated to the leucine.

[0267] Table 15: Catalytic results of rice HSL1 mutants towards different HPPD inhibitors

[0268] In Table 15, L204F / F298L / I335A refers to a mutant of rice HSL1 in which the 204th position of leucine is mutated to phenylalanine, the 298th position of phenylalanine is mutated to leucine, and the 335th position of isoleucine is mutated to alanine; L204F / F298L / I335W refers to a mutant of rice HSL1 in which the 204th position of leucine is mutated to phenylalanine, the 298th position of phenylalanine is mutated to leucine, and the 335th position of isoleucine is mutated to tryptophan; F140H / L204F / F298L / I335F refers to a mutant of rice HSL1 in which the 140th position of phenylalanine is mutated to histidine, the 204th position of leucine is mutated to phenylalanine, the 298th position of phenylalanine is mutated to leucine, and the 335th position of isoleucine is mutated to phenylalanine.

[0269] Table 16: Catalytic results of rice HSL2 mutants towards different HPPD inhibitors

[0270] In Table 16, T206F refers to a mutant in which the threonine at position 206 of rice HSL2 mutated to phenylalanine; T206F / F301L refers to a mutant in which the threonine at position 206 of rice HSL2 mutated to phenylalanine and the phenylalanine at position 301 mutated to leucine; T206F / F301L / Y339F refers to a mutant in which the threonine at position 206 of rice HSL2 mutated to phenylalanine, the phenylalanine at position 301 mutated to leucine, and the tyrosine at position 339 mutated to phenylalanine.

[0271] Table 17: Catalytic results of rice HSL4 mutants towards different HPPD inhibitors

[0272] In Table 17, Y142H refers to a mutant in which the tyrosine at position 142 of rice HSL4 mutated to histidine; Y142H / I207F refers to a mutant in which the tyrosine at position 142 of rice HSL4 mutated to histidine and the isoleucine at position 207 of rice HSL4 mutated to phenylalanine; Y142H / I207F / F301L refers to a mutant in which the tyrosine at position 142 of rice HSL4 mutated to histidine, the isoleucine at position 207 mutated to phenylalanine, and the phenylalanine at position 301 mutated to leucine.

[0273] Table 18: Catalytic results of wheat HSL2 mutants to different HPPD inhibitors

[0274] In Table 18, F285L refers to a mutant in which the phenylalanine at position 285 of wheat HSL2 is mutated to leucine; F285L / H322F refers to a mutant in which the phenylalanine at position 285 of wheat HSL2 is mutated to leucine and the histidine at position 322 is mutated to phenylalanine.

[0275] Table 19: Catalytic results of corn HSL1A mutants to different HPPD inhibitors

[0276] In Table 19, Q142H / R338F refers to a mutant in which the glutamine at position 142 of corn HSL1A mutated to histidine and the arginine at position 338 mutated to phenylalanine; Q142H / I207F / F301L / R338F refers to a mutant in which the glutamine at position 142 of corn HSL1A mutated to histidine, the isoleucine at position 207 mutated to phenylalanine, the phenylalanine at position 301 mutated to leucine, and the arginine at position 338 mutated to phenylalanine.

[0277] Table 20: Catalytic results of corn HSL1B mutants to different HPPD inhibitors

[0278] In Table 20, Q140H / Y205F refers to a mutant in which the glutamine at position 140 of corn HSL1B mutated to histidine and the tyrosine at position 205 mutated to phenylalanine; Q140H / Y205F / K336F refers to a mutant in which the glutamine at position 140 of corn HSL1B mutated to histidine, the tyrosine at position 205 mutated to phenylalanine, and the lysine at position 336 mutated to phenylalanine.

[0279] Table 21: Catalytic results of sorghum HSL1 mutants to different HPPD inhibitors

[0280] In Table 21, Q140H / Y205F refers to a mutant in which the glutamine at position 140 of sorghum HSL1 is mutated to histidine and the tyrosine at position 205 is mutated to phenylalanine; Q140H / Y205F / K336F refers to the amino acid sequence in which the glutamine at position 140 of sorghum HSL1 is mutated to histidine, the tyrosine at position 205 is mutated to phenylalanine, and the lysine at position 336 is mutated to phenylalanine.

[0281] The results of the above tests show that compared with the wild type (WT), the hydroxylation reaction activities of the mutant forms of rice HIS1, rice HSL1, rice HSL2, rice HSL4, wheat HSL2, maize HSL1A, maize HSL1B and sorghum HSL1 with HPPD inhibitors were significantly improved.

[0282] Specifically, the single-point mutation H141F of rice HIS1 has a catalytic efficiency of 31% for bracts and 37% for Y16550, which are 9% and 26% higher than those of the wild type, respectively; the double-point mutation H141F / F205L of rice HIS1 has a catalytic efficiency of 27% for bracts and 26% for Y16550, which are 5% and 15% higher than those of the wild type, respectively.

[0283] Specifically, the triple-point mutation L204F / F298L / I335A of rice HSL1 had a catalytic efficiency of 87% for BBC-OH, 72% for Y13287, 21% for Y18024, and 69% for Y16550, which were 38%, 72%, 21%, and 52% higher than those of the wild type, respectively.

[0284] The triple-point mutation L204F / F298L / I335W in rice HSL1 has a catalytic efficiency of 76% for mesotrione, 100% for BBC-OH, 64% for Y13287, 48% for Y18024, and 42% for Y16550, respectively, compared to the wild type.

[0285] The four-point mutation F140H / L204F / F298L / I335F of rice HSL1 has a catalytic efficiency of 85% for mesotrione, 100% for BBC-OH, 92% for Y13287, and 96% for Y18024, which are catalytic efficiencies increased by 45%, 51%, 92%, and 96%, respectively, compared with the wild type.

[0286] Specifically, the single point mutation T206F in rice HSL2 had a catalytic efficiency of 20% for mesotrione, 26% for BBC-OH, 8% for furazolidinone, 9% for Y13287, 16% for Y18024, and 11% for Y16550, which were 11%, 26%, 8%, 9%, 16%, and 6% higher than those of the wild type, respectively.

[0287] The double-point mutation T206F / F301L in rice HSL2 has a catalytic efficiency of 47% for mesotrione, 100% for BBC-OH, 25% for furazolidinone, 83% for Y13287, 30% for Y18024, and 17% for Y16550, respectively, compared with the wild type.

[0288] The triple-point mutations T206F / F301L / Y339F of rice HSL2 have a catalytic efficiency of 44% for mesotrione, 100% for BBC-OH, 21% for furansulfuron, 86% for Y13287, 27% for Y18024, 12% for bauhinia, and 16% for Y16550, which are catalytic efficiencies increased by 35%, 100%, 21%, 86%, 27%, 12%, and 11% compared with the wild type, respectively.

[0289] Specifically, the single-point mutation Y142H in rice HSL4 has a catalytic efficiency of 30% for furazolidinone, 75% for Y13287, and 8% for baculoyl sulfonamide, which are 13%, 2%, and 6% higher than those of the wild type, respectively.

[0290] The double-point mutation Y142H / I207F in rice HSL4 has a catalytic efficiency of 27% for furazolidinone, 76% for Y13287, and 5% for bauhinia, which are 10%, 3%, and 3% higher than those of the wild type, respectively.

[0291] The triple-point mutation Y142H / I207F / F301L in rice HSL4 has a catalytic efficiency of 22% for mesotrione and 4% for bauhinia, which are 1% and 2% higher than those of the wild type, respectively.

[0292] Specifically, the single-point mutation F285L in wheat HSL2 had a catalytic efficiency of 10% for BBC-OH, 32% for Y13287, and 34% for Y18024, which were 4%, 32%, and 34% higher than those of the wild type, respectively.

[0293] The double point mutation 285L / H322F of wheat HSL2 has a catalytic efficiency of 17% for BBC-OH, 12% for Y13287, and 9% for Y18024, which are 11%, 12%, and 9% higher than those of the wild type, respectively.

[0294] Specifically, the double-point mutation Q142H / R338F in maize HSL1A has a catalytic efficiency of 21% for mesotrione and 77% for furazolidinone, which are 8% and 28% higher than those of the wild type, respectively.

[0295] The catalytic efficiency of the four-point mutation Q142H / I207F / F301L / R338F in corn HSL1A for mesotrione is 15%, which is 2% higher than that of the wild type.

[0296] Specifically, the double-point mutation Q140H / Y205F in maize HSL1B has a catalytic efficiency of 18% for mesotrione, 100% for BBC-OH, and 9% for Y16550, which are 10%, 5%, and 3% higher than those of the wild type, respectively.

[0297] The triple-point mutation Q140H / Y205F / K336F in corn HSL1B has a catalytic efficiency of 19% for mesotrione and 100% for BBC-OH, which are 11% and 5% higher than those of the wild type, respectively.

[0298] Specifically, the double-point mutation Q140H / Y205F of sorghum HSL1 had a catalytic efficiency of 98% for BBC-OH, 7% for furazolidinone, and 7% for Y16550, which were 77%, 7%, and 7% higher than those of the wild type, respectively.

[0299] The triple-point mutation Q140H / Y205F / K336F of sorghum HSL1 has a catalytic efficiency of 26% for mesotrione, 100% for BBC-OH, 15% for furansulfuron, and 10% for Y16550, which are 6%, 79%, 15% and 10% higher than those of the wild type, respectively.

[0300] The rice HIS1, rice HSL1, rice HSL2, rice HSL4, wheat HSL2, sorghum HSL1, maize HSL1A, and maize HSL1 mutants provided herein have enhanced hydroxylation activity against HPPD inhibitors. In other words, the Fe(II) / α-ketoglutarate-dependent dioxygenase proteins provided herein are capable of detoxifying HPPD inhibitors. Therefore, the Fe(II) / α-ketoglutarate-dependent dioxygenases provided herein can be used to improve herbicide resistance in crops.

[0301] The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited thereto. Within the technical concept of the present invention, various simple variations of the technical solution of the present invention may be made, including combining the various technical features in any other appropriate manner. These simple variations and combinations should also be regarded as disclosed in the present invention and fall within the scope of protection of the present invention.

Claims

1. An Fe(Ⅱ) / α-ketoglutarate-dependent dioxygenase protein, characterized in that: Having an amino acid sequence selected from at least one of the following: (1) a first amino acid sequence derived from a mutation in at least one of positions 141 and 205 of the amino acid sequence shown in SEQ ID NO: 1; (2) a protein derived from the first amino acid sequence in which the first amino acid sequence is substituted, deleted or added with one or more amino acids, or a protein represented by an amino acid sequence with a tag connected to the amino terminus and / or carboxyl terminus of the first amino acid sequence; (3) a second amino acid sequence derived from mutations in at least three positions selected from the group consisting of positions 140, 204, 298, and 335 in the amino acid sequence of SEQ ID NO: 2; (4) a protein derived from the second amino acid sequence in which the second amino acid sequence is substituted, deleted or added with one or more amino acids, or a protein represented by the amino acid sequence with a tag connected to the amino terminus and / or carboxyl terminus of the second amino acid sequence; (5) a third amino acid sequence derived from a mutation in at least one of positions 206, 301 and 339 in the amino acid sequence of SEQ ID NO: 3; (6) a protein derived from the third amino acid sequence in which one or more amino acids are substituted, deleted or added to the third amino acid sequence, or a protein represented by the amino acid sequence with a tag connected to the amino terminus and / or carboxyl terminus of the third amino acid sequence; (7) a fourth amino acid sequence derived from a mutation in at least one of positions 142, 207 and 301 of the amino acid sequence as shown in SEQ ID NO: 4; (8) a protein derived from the fourth amino acid sequence in which one or more amino acids are substituted, deleted or added to the fourth amino acid sequence, or a protein represented by the amino acid sequence with a tag connected to the amino terminus and / or carboxyl terminus of the fourth amino acid sequence; (9) a fifth amino acid sequence derived from a mutation in at least one of positions 285 and 322 of the amino acid sequence as shown in SEQ ID NO: 5; (10) a protein derived from the fifth amino acid sequence in which one or more amino acids are substituted, deleted or added to the fifth amino acid sequence, or a protein represented by the amino acid sequence with a tag connected to the amino terminus and / or carboxyl terminus of the fifth amino acid sequence; (11) a sixth amino acid sequence derived from a mutation in at least one of positions 142, 207, 301 and 338 of the amino acid sequence as shown in SEQ ID NO: 6; (12) a protein derived from the sixth amino acid sequence in which one or more amino acids are substituted, deleted or added to the sixth amino acid sequence, or a protein represented by the amino acid sequence with a tag connected to the amino terminus and / or carboxyl terminus of the sixth amino acid sequence; (13) a seventh amino acid sequence derived from mutations in at least two of the amino acid positions selected from position 140, position 205, and position 336 in the amino acid sequence as shown in SEQ ID NO: 7; (14) a protein derived from the seventh amino acid sequence in which one or more amino acids are substituted, deleted or added to the seventh amino acid sequence, or a protein represented by the amino acid sequence with a tag connected to the amino terminus and / or carboxyl terminus of the seventh amino acid sequence; (15) an eighth amino acid sequence derived from mutations in at least two of the amino acid positions selected from position 140, position 205, and position 336 in the amino acid sequence as shown in SEQ ID NO: 8; (16) A protein derived from the eighth amino acid sequence in which one or more amino acids are substituted, deleted or added to the eighth amino acid sequence, or a protein represented by the amino acid sequence with a tag connected to the amino terminus and / or carboxyl terminus of the eighth amino acid sequence.

2. The Fe(II) / α-ketoglutarate-dependent dioxygenase protein according to claim 1, wherein The first amino acid sequence is an amino acid sequence selected from at least one of the following: (1a) an amino acid sequence derived from the amino acid sequence shown in SEQ ID NO: 1 after the histidine at position 141 is mutated to phenylalanine; (1b) an amino acid sequence derived from the amino acid sequence shown in SEQ ID NO: 1 in which the histidine at position 141 is mutated to phenylalanine and the phenylalanine at position 205 is mutated to leucine; Preferably, the first amino acid sequence is the amino acid sequence shown by H141F or H141F / F205L; Preferably, the second amino acid sequence is the following amino acid sequence: (2a) In the amino acid sequence shown in SEQ ID NO: 2, the phenylalanine at position 140 is mutated to histidine, and the leucine at position 204 is mutated to phenylalanine, an amino acid sequence derived from a mutation of the phenylalanine at position 298 to leucine and a mutation of the isoleucine at position 335 to phenylalanine; (2b) an amino acid sequence derived from the amino acid sequence of SEQ ID NO: 2 in which the leucine at position 204 is mutated to phenylalanine, the phenylalanine at position 298 is mutated to leucine, and the isoleucine at position 335 is mutated to alanine; (2c) an amino acid sequence derived from the amino acid sequence of SEQ ID NO: 2 in which the leucine at position 204 is mutated to phenylalanine, the phenylalanine at position 298 is mutated to leucine, and the isoleucine at position 335 is mutated to tryptophan; Preferably, the second amino acid sequence is at least one of the amino acid sequences shown in F140H / L204F / F298L / I335F, L204F / F298L / I335A, and L204F / F298L / I335W; preferably, the third amino acid sequence is the following amino acid sequence: (3a) an amino acid sequence derived from the amino acid sequence shown in SEQ ID NO: 3 in which the threonine at position 206 is mutated to phenylalanine; (3b) an amino acid sequence derived from the amino acid sequence shown in SEQ ID NO: 3 in which the threonine at position 206 is mutated to phenylalanine and the phenylalanine at position 301 is mutated to leucine; (3c) an amino acid sequence derived from the amino acid sequence shown in SEQ ID NO: 3 in which the threonine at position 206 is mutated to phenylalanine, the phenylalanine at position 301 is mutated to leucine, and the tyrosine at position 339 is mutated to phenylalanine; Preferably, the third amino acid sequence is at least one of the amino acid sequences shown by T206F, T206F / F301L, and T206F / F301L / Y339F; Preferably, the fourth amino acid sequence is the following amino acid sequence: (4a) an amino acid sequence derived from the amino acid sequence of SEQ ID NO: 4 in which tyrosine at position 142 is mutated to histidine; (4b) an amino acid sequence derived from the amino acid sequence shown in SEQ ID NO: 4 in which the tyrosine at position 142 is mutated to histidine and the isoleucine at position 207 is mutated to phenylalanine; (4c) an amino acid sequence derived from the amino acid sequence of SEQ ID NO: 4 in which the tyrosine at position 142 is mutated to histidine, the isoleucine at position 207 is mutated to phenylalanine, and the phenylalanine at position 301 is mutated to leucine; Preferably, the fourth amino acid sequence is at least one of the amino acid sequences shown by Y142H, Y142H / I207F, and Y142H / I207F / F301L; Preferably, the fifth amino acid sequence is the following amino acid sequence: (5a) an amino acid sequence derived from the amino acid sequence of SEQ ID NO: 5 in which the phenylalanine at position 285 is mutated to leucine; (5b) an amino acid sequence derived from the amino acid sequence shown in SEQ ID NO: 5 in which the phenylalanine at position 285 is mutated to leucine and the histidine at position 322 is mutated to phenylalanine; Preferably, the fifth amino acid sequence is at least one of the amino acid sequences shown by F285L and F285L / H322F; Preferably, the sixth amino acid sequence is the following amino acid sequence: (6a) an amino acid sequence derived from the amino acid sequence shown in SEQ ID NO: 6 where glutamine at position 142 is mutated to histidine; (6b) an amino acid sequence derived from the amino acid sequence shown in SEQ ID NO: 6 in which the glutamine at position 142 is mutated to histidine and the isoleucine at position 207 is mutated to phenylalanine; (6c) an amino acid sequence derived from the amino acid sequence of SEQ ID NO: 6 in which the glutamine at position 142 is mutated to histidine, the isoleucine at position 207 is mutated to phenylalanine, and the phenylalanine at position 301 is mutated to leucine; (6d) an amino acid sequence derived from the amino acid sequence of SEQ ID NO:6 in which the glutamine at position 142 is mutated to histidine, the isoleucine at position 207 is mutated to phenylalanine, the phenylalanine at position 301 is mutated to leucine, and the arginine at position 338 is mutated to phenylalanine; (6e) an amino acid sequence derived from the amino acid sequence of SEQ ID NO: 6 in which the glutamine at position 142 is mutated to histidine and the arginine at position 338 is mutated to phenylalanine; Preferably, the sixth amino acid sequence is at least one of the amino acid sequences shown in Q142H, Q142H / I207F, Q142H / I207F / F301L, Q142H / I207F / F301L / R338F, and Q142H / R338F; Preferably, the seventh amino acid sequence is the following amino acid sequence: (7a) an amino acid sequence derived from the amino acid sequence of SEQ ID NO: 7 in which the glutamine at position 140 is mutated to histidine and the tyrosine at position 205 is mutated to phenylalanine; (7b) In the amino acid sequence shown in SEQ ID NO: 7, the glutamine at position 140 is mutated to histidine, and the tyrosine at position 205 is mutated to The amino acid sequence derived from the mutation of phenylalanine and lysine at position 336 to phenylalanine; Preferably, the seventh amino acid sequence is at least one of the amino acid sequences shown by Q140H / Y205F and Q140H / Y205F / K336F; Preferably, the eighth amino acid sequence is the following amino acid sequence: (8a) an amino acid sequence derived from the amino acid sequence of SEQ ID NO: 8 in which the glutamine at position 140 is mutated to histidine and the tyrosine at position 205 is mutated to phenylalanine; (8b) an amino acid sequence derived from the amino acid sequence of SEQ ID NO: 8 in which glutamine at position 140 is mutated to histidine, tyrosine at position 205 is mutated to phenylalanine, and lysine at position 336 is mutated to phenylalanine; Preferably, the eighth amino acid sequence is at least one of the amino acid sequences shown by Q140H / Y205F and Q140H / Y205F / K336F.

3. A gene encoding a Fe(II) / α-ketoglutarate-dependent dioxygenase protein, characterized in that: The nucleotide sequence of the gene is a nucleotide sequence capable of encoding the amino acid sequence of the Fe(II) / α-ketoglutarate-dependent dioxygenase protein according to claim 1 or 2; Preferably, the nucleotide sequence of the gene is at least one nucleotide sequence selected from the following: (1) the nucleotide sequence shown in SEQ ID NO: 11; (2) a nucleotide sequence that is at least 90% identical, preferably at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 11; (3) the nucleotide sequence shown in SEQ ID NO:21; (4) a nucleotide sequence that is at least 90% identical, preferably at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 21; (5) the nucleotide sequence shown in SEQ ID NO:31; (6) a nucleotide sequence that is at least 90% identical, preferably at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 31; (7) the nucleotide sequence shown in SEQ ID NO:41; (8) a nucleotide sequence that is at least 90% identical, preferably at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO:41; (9) the nucleotide sequence shown in SEQ ID NO:51; (10) a nucleotide sequence that is at least 90% identical, preferably at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO:51; (11) the nucleotide sequence shown in SEQ ID NO:61; (12) a nucleotide sequence that is at least 90% identical to SEQ ID NO: 61, preferably at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical; (13) the nucleotide sequence shown in SEQ ID NO:71; (14) a nucleotide sequence that is at least 90% identical to SEQ ID NO: 71, preferably at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical; (15) the nucleotide sequence shown in SEQ ID NO:81; (16) having at least 90% identity, preferably at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 81 nucleotide sequence.

4. A recombinant vector, characterized in that: The recombinant vector contains the gene according to claim 3.

5. A transgenic cell, characterized in that: The transgenic cell contains the gene according to claim 3; Preferably, the transgenic cell is a prokaryotic cell.

6. A composition, characterized in that The composition contains the Fe(II) / α-ketoglutarate-dependent dioxygenase protein according to claim 1 or 2.

7. Use of at least one of the Fe(II) / α-ketoglutarate-dependent dioxygenase protein according to claim 1 or 2, the gene according to claim 3, the recombinant vector according to claim 4, and the transgenic cell according to claim 5 in improving crop herbicide resistance; Preferably, the herbicide is a HPPD inhibitor; Preferably, the herbicide is at least one of a triketone compound, a pyrazole compound, an isoxazole compound, a diketonitrile compound and a heterocyclic amide compound; More preferably, the herbicide is at least one of mesotrione, cypermethrin, furansulfuron, bicyclonazole, quinclorac, methylquinclorac, Y18024, Y16550 and benzylpyrazone.

8. A method for improving crop herbicide resistance, characterized in that: The method comprises: transferring the recombinant vector of claim 4 into a target plant, so that the target plant expresses the Fe(II) / α-ketoglutarate-dependent dioxygenase protein of claim 1 or 2 to obtain resistance to herbicides.

9. A method for improving crop herbicide resistance, characterized in that: The method comprises: transferring the recombinant vector in a mutant crop containing the recombinant vector according to claim 4 into a target plant by hybridization, breeding or backcrossing, so that the target plant expresses the Fe(II) / α-ketoglutarate-dependent dioxygenase protein according to claim 1 or 2 to obtain resistance to herbicides.

10. A method for improving crop herbicide resistance, characterized in that: The method comprises: modifying the HIS1 / HSL gene of the target plant by the CRISPR / Cas gene editing method, so that the target plant expresses the Fe(II) / α-ketoglutarate-dependent dioxygenase protein according to claim 1 or 2, so as to obtain resistance to the herbicide.

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